Inhibition of secreted phospholipase A2 by neuron survival and anti-inflammatory peptide CHEC-9
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The nonapeptide CHEC-9 (CHEASAAQC), a putative inhibitor of secreted phospholipase A2 (sPLA2), has been shown previously to inhibit neuron death and aspects of the inflammatory response following systemic treatment of rats with cerebral cortex lesions. In this study, the properties of CHEC-9 inhibition of sPLA2 enzymes were investigated, using a venom-derived sPLA2 group I and the plasma of rats and humans as the sources of enzyme activity. The results highlight the advantages of inhibitors with uncompetitive properties for inflammatory disorders including those resulting in degeneration of neurons.
Samples of enzyme and plasma were reacted with 1-Palmitoyl-2-Pyrenedecanoyl Phosphatidylcholine, a sPLA2 substrate that forms phospholipid vesicles in aqueous solutions. Some of the plasma samples were collected from restrained peptide-treated rats in order to confirm the validity of the in vitro assays for extrapolation to in vivo effects of the peptide. The enzyme reactions were analyzed in terms of well-studied relationships between the degree of inhibition and the concentrations of different reactants. We also examined interactions between different components of the reaction mixture on native polyacrylamide gels.
In all cases, the peptide showed the properties of an uncompetitive (or anti-competitive) enzyme inhibitor with Ki values less than 100 nanomolar. The electrophoresis experiments suggested CHEC-9 modifies the binding properties of the enzyme only in the presence of substrate, consistent with its classification as an uncompetitive inhibitor. Both the in vitro observations and the analysis of plasma samples from restrained rats injected with peptide suggest the efficacy of the peptide increases under conditions of high enzyme activity.
Modeling studies by others have shown that uncompetitive inhibitors may be optimal for enzyme inhibition therapy because, unlike competitive inhibitors, they are not rendered ineffective by the accumulation of unmodified substrate. Such conditions likely apply to several instances of neuroinflammation where there are cascading increases in sPLA2s and their substrates, both systemically and in the CNS. Thus, the present results may explain the efficacy of CHEC-9 in vivo.
KeywordsSubstrate Concentration Secrete Phospholipase Uncompetitive Inhibitor Increase Substrate Concentration sPLA2 Activity
Pro-inflammatory enzymes and cytokines are increasingly attractive as therapeutic targets for a variety of inflammatory diseases and for the inflammatory component of neurodegenerative disorders. The 14–18 kD secreted phospholipase A2s (sPLA2s) are of interest in this regard because of their accessibility in the circulation and because local and systemic elevation of sPLA2s are associated with most forms of inflammation [1, 2, 3, 4, 5]. The secreted isoforms are part of a growing family of PLA2 enzymes whose activity leads to the production of several potent mediators of inflammation. Increased levels of extracellular sPLA2s have been detected in the plasma of patients affected by systemic inflammatory diseases such as acute pancreatitis, septic shock, extensive burns, and autoimmune diseases. The enzymes are also accumulated in inflammatory fluids such as the synovial fluid of patients with rheumatoid arthritis, the bronchoalveolar lavage of patients with bronchial asthma, and the nasal secretions of patients with allergic rhinitis. More recent experimental studies suggest sPLA2s are involved in traumatic and autoimmune precipitated neurodegeneration, and thus these enzymes are also a potential target for treatment of nervous system disorders.) [6, 7, 33].
CHEC-9 is a putative sPLA2 inhibitor that has recently been identified as an internal fragment of the survival-promoting, anti-inflammatory polypeptide DSEP/Dermcidin/PIF [6, 8, 9, 10, 11, 12]. Following cerebral cortex lesions, a subcutaneous injection of CHEC-9 results in anti-inflammatory and neuron survival effects that last for at least 4 days, an effect due in part to an interruption of the inflammatory cascade that follows damage to the CNS. Given the efficacy of CHEC-9, the present study was undertaken to investigate CHEC-9 inhibition of sPLA2 activity in detail. The results suggest that CHEC-9 has several characteristics of an uncompetitive (or anti-competitive) sPLA2 inhibitor even when tested ex vivo with a chemically complex fluid like plasma. These properties are likely to be especially advantageous under conditions of inflammation and associated oxidative stress, and therefore are consistent with the peptide's performance in vivo.
Sources and preparation of sPLA2
Purified secreted phospholipase A2 group I from the venom of the Mozambique cobra (Naja mossambica) was obtained from Sigma. Blood was obtained from the trunk of 20 female Sprague Dawley rats (200–250 g) after decapitation, and by venipunture of 14 healthy adult humans of both sexes. Blood samples from 8 additional rats were collected following subcutaneous injections of 100 μg CHEC-9 or DMEM vehicle. These rats were placed in a standard rat restrainer during the collection period and the samples obtained via a tail nick. All specific procedures of this study were approved by both the Institutional Animal Care and Use Committee and by Institutional Review Board of Drexel University College of Medicine. Blood samples were treated with citrate-phosphate-dextrose anticoagulant (1:10, Sigma), and plasma prepared by centrifugation, before freezing at -80° until used in the enzyme assays. For the ex vivo studies, individual plasma samples were pooled from 3–7 rats or 3–5 humans.
Enzyme assays were conducted at ambient temperature (22–25°) using a Victor 3 fluorescent reader (Perkin Elmer, Nutley NJ). The substrate was 1-Palmitoyl-2-Pyrenedecanoyl Phosphatidylcholine ("10-pyrene", Caymen Chemical, Ann Arbor MI) a substrate for all calcium dependent PLA2s with the exception of cPLA2 and PAF-AH. The substrate (supplied in chloroform) was dried under a nitrogen stream, quickly dissolved in ethanol, and stored at -20° prior to use. Substrate solutions were prepared in reaction buffer consisting of 50 mM tris (pH = 7.4), 0.1 M NaCl, 2 mM CaCl2, 0.25% fatty acid-free albumin (Sigma) and the CHEC-9 peptide at the indicated concentrations. CHEC-9 (CHEASAAQC) was synthesized by Celtek, (Nashville, TN), purified and cross-linked as described previously , and aliquots stored in tris buffer or DMEM vehicles at -80°. The 10-pyrene substrate forms phospholipids vesicles in aqueous solutions , and upon hydrolysis, releases 10-pyrenyldecanoic acid. This product is fluorescent in the presence of albumin and was measured at 350 nm excitation, 405 nM emission. Plasma samples were 20% final concentration in the reaction mixture, and all enzyme reactions were initiated with the addition of the substrate solution to the sPLA2 containing samples. Kinetic parameters including the properties of CHEC-9 were determined by measuring the initial velocities (Vo) of enzyme reactions (within 2 minutes of initiation). For experiments in which active sPLA2 enzyme concentration was measured in plasma samples from peptide-treated rats, we used a single substrate concentration and measured the steady-state rate of the enzyme reaction for 30 minutes. This rate is proportional to the concentration of active enzyme in the plasma if product formation during this period is linear with respect to time (see Fig. 6). In most experiments, relative fluorescent units (RFU) were converted to product concentration using a pyrenyldecanoic acid standard curve (Molecular Probes, Eugene, OR). For plasma, the background fluorescence of the plasma was not subtracted, but this did not effect the velocity measurements. Individual reactions were carried out in duplicate or triplicate and kinetic curves were produced using 5–6 substrate concentrations, with or without peptide, reacted simultaneously. Representative Lineweaver-Burke plots and nonlinear regression analyses of reactions using multiple peptide and substrate concentrations are presented in Results. Individual experiments were repeated 5 or more times with the same result, i.e., the direction of change of Km and Vmax was the same following inhibitor treatment, and Ki was less than 100 nM. Km and Vmax and r2 were determined with regression software (Prism) from Graphpad (San Diego, CA).
Identification of inhibitor properties
The characteristics of CHEC-9 inhibition were determined using both classical characterization of inhibitor types, i.e., the direction of changes in Km and Vmax, and more recent reports that derive consistent relationships between the extent of enzyme inhibition and substrate concentration [14, 15, 16, 17]. The analyses of Geng  and Whitely were particularly useful for the present studies because they allowed classification of CHEC-9 as well as calculation of the conventional inhibition constants for the various enzyme sources. We have repeated, combined, and rearranged some of their equations below in order to show the calculations used in the present experiments.
KiNR is the apparent inhibition constant based on inhibition degree and is independent of inhibitor classification . This value varies predictably with substrate concentration for different inhibitor types. It is defined as:
K iNR = [I]·R/(1-R), where [I] = inhibitor concentration, and
R = velocity+inhibitor/velocity-inhibitor.
This equation can be rewritten as:
K iNR = [I]·velocity+inhibitor/(velocity-inhibitor - velocity+inhibitor) for expressing KiNR in terms of measured velocities. For the experiments in which the Michaelis constant Km was reliably estimated by nonlinear regression, Ki for an uncompetitive inhibitor was calculated from the following:
R = 1/(1 + ([I]/Ki·(1+(Km/[S])), where [S] = concentration of substrate.
This rearranges to: Ki = (R/(1-R))·[I]/(1+(Km/[S])), or, in terms of KiNR:
Ki = KiNR/(1+(Km/[S])).
Polyacrylamide gel electrophoresis
Polyacrylamide gradient gels (5–15%) were run with and without SDS or reducing agents using sPLA2 group I alone or after mixing the enzyme with different combinations of the components of the reaction mixture (or their solvents). Samples prepared for the native gels were 50 μl containing 26 μM sPLA2, 40 μM CHEC-9, 560 μM 1-Palmitoyl-2-Pyrenedecanoyl Phosphatidylcholine (substrate), and 2 mM CaCl2 in 20 mM tris buffer (pH = 7.4), incubated together at room temperature for 30 min. After incubation, the samples were evaporated to 20 μl. The samples were then mixed with sample buffer containing only glycerol and bromophenol blue in 0.1 M tris (pH = 6.8) and loaded onto the gels. Following electrophoresis, the gels were washed, fixed, and stained with silver reagent according to conventional methods. The native gel experiment, using different reactant combinations run side by side (Fig. 3), was repeated four times with the same result.
sPLA2 group I
Kinetic Parameters at Different Inhibitor Concentrations
Group I sPLA2
Ki = 4.02 ± 1.56 nM
Human Plasma sPLA2
Polyacrylamide gel electrophoresis
In the electrophoresis experiments the concentrations of the participants in the reaction were scaled up so the migration of the enzyme on native gels could be observed following incubation with various reactants. We first confirmed that the sPLA2 used in these experiments was a single species after electrophoresis using conventional buffers with or without SDS and reducing reagents (not shown). The native gels had high argentophilic background but a single discrete sPLA2 band was still observed when the enzyme was pre-incubated in only the modified tris-calcium reaction buffer (without substrate or peptide, lane 2, Fig. 3). The same band appeared when either substrate or peptide alone was added to this mixture (lanes 3 and 4 arrowhead), although in the case of the former, a large, irregular, and intensely argentophilic band also appeared nearer the top of the gels, presumably representing a product or intermediate in the enzyme-substrate reaction. This same large band was also apparent when CHEC-9, enzyme and substrate were all present in the sample. However, the sPLA2 band was absent or at least dramatically reduced with CHEC-9 present (lane 1). Since the sPLA2 disappears or is diminished only in the presence of substrate and CHEC-9, it is suggested that peptide, substrate, and enzyme formed a complex that precluded the migration of the enzyme to its typical position in native gels. This result was consistent with the properties of an uncompetitive inhibitor.
In vivo experiments
Competitive versus uncompetitive inhibitors
Studies of enzyme inhibitors in open systems have suggested that competitive inhibitors, still a major focus of therapeutic drug design, have inherent limitations that may compromise their efficacy . These limitations involve the accumulation of unmodified substrate, a natural result of blocking of the enzyme binding to substrate by the inhibitor. The accumulated substrate will eventually compete successfully for the enzyme overcoming the inhibition. These limitations are expected to be especially apparent in situations of both acute and persistent inflammation, the conditions that are amenable to sPLA2 inhibition therapy. Even a more or less localized inflammatory lesion is accompanied by both a localized and systemic sPLA2 response as has been demonstrated for a variety of disorders, including following nervous system lesions (see Introduction). Inflammation and associated oxidative stress will also increase levels of sPLA2 substrates including, (but not exclusively), acidic phospholipids expressed at the cell surface , phosphatidic acid released by activated immune cells[20, 21], and oxidized low density lipoproteins, components of the phospholipid targets in circulating lipoproteins [22, 23]. Successful enzyme inhibition therapy, introduced systemically, must therefore overcome widespread elevations in enzyme and available substrates. The uncompetitive sPLA2 inhibitor may have an advantage under such conditions, since this increased activity may actually favor the enzyme inhibition. Therefore, competitive sPLA2 inhibitors, even those displaying potent inhibition in vitro, may have short-lived effects in vivo depending on the level and persistence of the inflammatory response. It is possible that this limitation contributed to the lack of in vivo efficacy of a number of potent small molecule competitive sPLA2 inhibitors developed commercially and abandoned , as well as to the poor performance of competitive sPLA2 inhibitors in recent clinical trials [4, 24, 25]. It should be noted however that these inhibitors were not tested in models of neurodegenerative disease.
Neurodegeneration and PLA2 isoforms
The survival-promoting and anti-inflammatory effects of CHEC-9 are most readily explained by the inhibition of PLA2 activity, either sPLA2s directly, or cytosolic PLA2 which may be regulated by sPLA2s during oxidative stress [19, 26, 27]. The activity of the PLA2 enzymes has been associated with cell degeneration in many systems including the nervous system [28, 29]. Furthermore, microglia and macrophages may depend on PLA2 activity for cell killing [30, 31]. At present, the PLA2 isoforms targeted by CHEC-9, where in vivo they are targeted, and their relevance to particular neurodegenerative disorders is unknown. We have emphasized ex vivo experiments with plasma and sPLA2 activity in plasma after in vivo exposure to CHEC-9 because these fluids, while complex in terms of the number of active PLA2 isoforms present [2, 22], likely contribute to the systemic component of neuroinflammatory disorders. In fact, the efficacy of CHEC-9 in plasma, as well as with a venom-derived sPLA2, indicates broad specificity of the peptide, which may also contribute to its effectiveness in vivo. The nonenzymatic functions of PLA2 enzymes may also contribute to the pathophysiology of neurodegenerative diseases, and CHEC-9 could also influence these activities at the same time or independently of enzyme inhibition [2, 32].
The contribution of sPLA2 enzyme activity to inflammatory and degenerative disorders of the nervous system is increasingly appreciated. Given the nature of inflammatory stimuli and of the inflammatory cascade, inhibitors of enzyme activity with uncompetitive properties may be optimal for therapeutic intervention, since their efficacy is increased under conditions of escalating enzyme activity.
This work was supported by a grant from the Amyotrophic Lateral Sclerosis Association.
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